This proposal focuses on studying RNA silencing using an Agrobacterium-mediated transient assay system. RNA silencing was recently identified as an adaptive, antiviral host defense response in plants. RNA silencing limits the extent of a virus infection through sequence-specific cleavage of viral RNA. RNA silencing in plants is closely related to the process of RNA interference in animals, and quelling in fungi. Through genetic and biochemical analysis, the molecular basis for RNA silencing is partially understood. A key component of the RNA silencing pathway is the host RdRp. The RdRp is proposed to be important for the initiation and perhaps maintenance of the RNA silencing signal. A series of experiments to test the role of the RdRp in the two RNA silencing induction pathways are proposed. Many viruses encode RNA silencing suppressors. P1/HC-Pro, a silencing suppressor of potyviruses, will be used in the transient assay to further test its role in silencing suppression. Introducing a combination of components and effectors of the silencing pathway will allow for a more detailed understanding of the initiation, maintenance, and signaling stages of RNA silencing.